The acquired immunodeficiency syndrome (AIDS) is a sexually transmitted disease that can also be transmitted through contaminated blood or blood products. It is caused by human immunodeficiency virus (HIV, previously called HTLV-III) which infects and is latently harboured in T4-lymphocytes and monocytes (Wong-Staal, F. and Gallo, R. C.: Human T-lymphotropic retroviruses. Nature 317:395-403, 1985). The chronically HIV-infected individual may in turn transmit HIV, most often by sexual contact. During infection, the immune response deteriorates and AIDS develops in 50-70% of the cases. The fate for the remainder of infected persons is not yet known. When AIDS develops it is lethal and characterised by opportunistic infection, Kaposi sarcoma, other tumors and/or neurological disease.
To diagnose an HIV infection, virus is isolated or the antibody response is measured. Virus isolation is successful in 30-50% of asymptomatic HIV infected persons, and in 90-100% of patients who developed AIDS. The antibody response is composed of immunoglobulins directed to the various structural and enzymatic components of HIV. They include the virus envelope proteins glycoprotein (gp)120, the transmembrane protein gp41 and their precursor gp160, the interior structural group antigens p24, 17 and 7/9 and their precursor p 55, the enzymes reverse transcriptase (RT) p65/51 and endonuclease p32 and protease. Antibodies to proteins of the regulatory regions of the HIV genome also develop. The correct identification of an HIV-infected person depends on the type(s) of immunoglobulin (Ig) he produces and on the correct composition of antigens used in the immunoassay.
One common way to establish a diagnosis through antibody detection is to screen serum samples by enzyme-linked immunosorbent assay (ELISA) (Sarngadharan, M. G., Popovic, M., Bruch, L., Schupbach, J. and Gallo, R. C.: Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 224:506-508, 1984; Schupbach, J., Haller, O., Vogt, M., Luthy, R., Joller, H., Oelz, O., Popovic, M., Sarngadharan, M. G. and Gallo, R. C.: Antibodies to HTLV-III in Swiss patients with AIDS and pre-AIDS and in groups at risk for AIDS. The New Engl. J. Med. 312:265-270, 1985). Wells of microplates coated with viral antigens are reacted with the serum samples under investigation, washed, and antihuman Ig added. The latter reagent is labelled with an enzyme. After washing, the enzyme labelled antihuman Ig remains only if specific antiviral Ig was present in the serum sample. It is visualized by addition of a substrate for the enzyme and the color reaction quantified in a spectrophotometer. To verify this positive ELISA reaction, the serum sample is then added to e.g. Western blots which contain electrophoretically separated virus subcomponents. An immunoreaction on the Western blot will show whether Ig is reactive with the bands characteristic of viral subcomponents. Usually two different and characteristic bands are required to establish a definite diagnosis of HIV antibody.
The reliability of HIV antibody detection is dependent on the reagents of the ELISA plate. Lysates of infected cells may contain cellular contaminants, causing false positive serological reactions (Saag, M. S. and Britz, J.: Asymptomatic blood donor with a false positive HTLV-III western blot. The New Engl. J. Med. 314:118, 1985, Martin, P. W., Burger, D. R., Caouette, S. and Goldstein, A. S.: Importance of confirmatory tests after strongly positive HTLV-III screening tests. The New Engl. J. Med. 314:1577, 1986). Viral lysates usually have an over-representation of gag proteins and less envelope proteins which may cause false negative reactions. In addition to native viral lysates, recombinant-produced polypeptides have been produced (Ghrayeb, J., Kato. I., McKinney, S., Huang, J. J., Chanda, P. K., Ho. D. D., Sarngadharan, M. G., Chang, T. W. and Chang, N. T.: Human T-cell lymphotropic virus type III (HTLV-III) core antigens: synthesis in Escherichia coli and immunoreactivity with human sera. DNA 5:93-99, 1986; Chang, T. W., Kato, I., McKinney, S., Chanda, P., Barone, A. D., Wong-Staal, F., Gallo, R. C. and Chang, N. T.: Detection of antibodies to human T-cell lymphotropic virus-III (HTLV-III) with an immuno-assay employing a recombinant Escheria coli-derived viral antigenic peptide. Bio/Technology 3:905-909, 1985). The specificity and sensitivity of these assays are good, but not 100% (Chang, T. W. et al. ibid; Deinhardt, F., Eberle, J. and Gurtler, L.: Sensitivity and specificity of eight commercial and one recombinant anti-HIV ELISA tests. Lancet: 40, 1987).
The envelope protein gp120 varies between isolates (Wong-Staal, F. et al, ibid; Weiss, R. A., Clapham, P. R., Weber, J. N., Dalgleish, A. G., Lasky, L. A. and Berman, P. W.: Variable and conserved neutralization antigens of human immunodeficiency virus. Nature 324:572-575, 1986) and consequently peptides from them are not usually recommendable as ELISA antigens. Antibodies to gp41 have instead been considered the most sensitive diagnostic criterion of HIV infection (Sarngadharan, M. G., et al, ibid; Schupbach, J., et al, ibid). gp41 contains regions that are well conserved between various isolates. Antibodies to p24 disappear with progressive disease (Lange, J. M. A., Paul, D. A., Huisman, H. G., de Wolf, F., van der Berg, H., Coutinho, R. A., Danner, S. A., van der Noordaa, J. and Goudsmit, J.: Persistent HIV antigenaemia and decline of HIV core antibodies associated with transition to AIDS. British. Med. J. 293:1459-1461, 1986) but are the ones which appear early. Regarding known antibody responses, a combination of measuring anti p24 and anti gp41 should therefore be the most reliable for all circumstances.
Performing Western blot is time consuming and expensive. To produce reliable antigens for use in ELISA seems preferable. No single assay has so far obtained 100% specificity and sensitivity (Deinhardt, F., et al, ibid; Lange, J. M. A., et al, ibid).
Recently, Gnann, J. W., et al (Journal of Virology, Aug. 1987, p. 2639-2641) identified a 12-amino-acid peptide derived from gp41 of HIV, that was recognized by 100% of the sera from HIV-infected sera. The described peptide contained two cystein residues, and the authors stated that they provide evidence that disulfide bond formation plays a key role in the antigenic conformation of the epitope. However, no such evidence was presented. The authors assumed that the peptide had cyclized spontaneously, and they attempted to prove it by reducing the alleged disulfide bond, but they failed. Instead they synthesized another 12-amino-acid peptide in which they had substituted serine for one of the cysteines, and when this was tested it reacted with only 2 of 22 HIV-positive test sera. The authors conclude that the presence of both cysteine residues is essential for the antigenic conformation of the epitope, possibly via formation of disulfide bonds. They have not proved that they had a disulfide bond in the investigated peptide, what they have done is to show that cysteine is better than serine in the specified position of said peptide. The existence of two cysteines in a peptide is not equivalent to a cyclic structure. The cyclization demands a defined chemical step, namely oxidation, and the result can be method dependent. If spontaneous cyclization occurs it is often incomplete and accompanied by polymerization.
In primary HIV disease, antibody tests may be negative during a period (Marlink, R. G., Allan, J. S., McLane, M. F., Essex, M., Anderson, K. C. and Groopman, J. E.: Low sensitivity of ELISA testing in early HIV Infection. The New Engl. J. Med. 315:1549, 1986).
The development of a single rapid, sensitive and specific assay for detecting antibodies to HIV, also at an early stage of infection, is most desirable.